e IF 5 B employs a novel domain release mechanism to catalyze ribosomal subunit joining

e IF 5 B is a eukaryal translational GTP ase that catalyzes ribosomal subunit joining to form elongation‐competent ribosomes. Despite its central role in protein synthesis, the mechanistic details that govern the function of e IF 5 B or its archaeal and bacterial ( IF 2) orthologs remained unclear. Here, we present six high‐resolution crystal structures of e IF 5 B in its apo, GDP ‐ and GTP ‐bound form that, together with an analysis of the thermodynamics of nucleotide binding, provide a detailed picture of the entire nucleotide cycle performed by e IF 5 B . Our data show that GTP binding induces significant conformational changes in the two conserved switch regions of the G domain, resulting in the reorganization of the GTP ase center. These rearrangements are accompanied by the rotation of domain II relative to the G domain and release of domain III from its stable contacts with switch 2, causing an increased intrinsic flexibility in the free GTP ‐bound e IF 5 B . Based on these data, we propose a novel domain release mechanism for e IF 5 B / IF 2 activation that explains how e IF 5 B and IF 2 fulfill their catalytic role during ribosomal subunit joining.